DNA Polymerases a! and 6 Are Immunologically and Structurally Distinct

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 264, No. 10, Ienue of April 5, pp. 5924-5928,1989 Printed in U.S.A.

Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc

DNA Polymerases a! and 6 Are Immunologically andStructurally Distinct* (Received for publication, October 18, 1988)

Scott W. WongSj, Juhani SyvaojaTl, Cheng-Keat Tan(1,Kathleen M. Downey 11, Antero G . SoII, Stuart Linnq, and Teresa S.-F. Wang$** From the $Laboratoryof Experimental Oncology, Department of Pathology, Stanford University Medical School, Stanford, California 94305, the YDepartment of Biochemistry, University of California, Berkeley, California 94720, and the 11Department of Medicine and Biochemistry and Center for Blood Diseases, University of MiamiSchool of Medicine, Miami, Florida 33101

The relationship between DNA polymerases a and 6 are evaluated immunologically bymonoclonal antibody specifically against DNA polymerase a and murine polyclonal antiserum against calf thymus DNA polymerase 6. DNA polymerases a and 6 are found to be immunologically distinct. The structural relationship between the proliferating cell nuclear antigen (PCNA)-dependent calf DNA polymerase 6 and DNA polymerase a from human and calf was analyzed by two-dimensional tryptic peptide mapping of the catalytic polypeptides. The results demonstrate that the catalytic polypeptides of the PCNA-dependent calf polymerase 6 and DNA polymerase a are distinct, unrelated, and do not share any common structural determinants. The immunological and structural relationship between a recently identified PCNA-independent form of DNA polymerase 6 fromHeLa cells was also assessed. This PCNA-independent human polymerase 6 was found to be immunologically unrelated to human polymerase a butto share some immunological and structural determinants with the PCNA-dependent calf thymus polymerase 6.

A prerequisite of elucidating the mechanism of eukaryotic DNA replication is the identification and characterization of the key protein participants involved in this complex process. DNA polymerase a is generally accepted as a key polymerase involved in chromosomal DNA replication. Results from numerous studies supporting this claim are: (a)DNA polymerase a gene expression positively correlates with activation of cell proliferation and cellular transformation at both transcriptional and post-transcriptionallevels (Wong et al., 1988; Wahl et al., 1988); ( b ) several inhibitors of DNA polymerase a also inhibit DNA synthesis in uiuo (Fry and Loeb, 1986); ( c ) a temperature-sensitive mutant for DNA synthesis has been identified as a DNA polymerase a mutant (Murakami et al., 1985); ( d ) monoclonal antibodies specifically against DNA polymerase a inhibit DNA synthesis inpermeabilized cells or * This research was supported in part by Grants CA14835 (to T. S.-F. W.), DK26206 (to A. G. S.), and GM30415 (to S. L.)from the National Institutes of Health and a gift from the Donald E. and Delia B. Baxter Fund (to T. S.-F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Postdoctoral fellow supported by National Institutes of Health Training Grant CA09151. ** To whom correspondence should be addressed.

when microinjected into nuclei (Tanaka et al., 1982; Miller et al., 1985, 1986); ( e ) polymerase a is required for in uitro viral SV40 DNAreplication, which is a model system for eukaryotic DNA replication (Li and Kelly, 1984, 1985; Murakami et al., 1986; Stillman and Gluzman, 1985); and ( f ) most recently, the primary protein sequence of DNA polymerase a deduced from the cDNA sequence has been found to contain regions having striking similarity to both prokaryotic and eukaryotic replicative DNA polymerases (Wong et al., 1988). Another mammalian DNA polymerase, polymerase 6, has also implicated in theDNA replication process by its presence in proliferative tissuesand cells and by its sensitivity to replication inhibitors (Byrnes et al., 1976;Lee et al., 1981, 1985; Dresler and Frattini, 1986; Decker et al., 1987; Hammond et al., 1987;Lee and Toomey, 1987). An auxiliary protein that allows DNA polymerase 6, but not DNA polymerase a,to utilize primed long single-stranded template in a processive manner and also enables polymerase 6 to catalyze limited strand-displacementsynthesis on (dT)lp..la-primed, poly(A)-tailed pBR322 was identified (Tan et al., 1986; Downey et al., 1988). This auxiliary protein was subsequently demonstrated to be identified to proliferating cell nuclear antigen (PCNA),’ also known as cyclin (Bravo and Celis, 1980; Mathews et al., 1984; Bravo, 1986; Bravo et al., 1987; Prelich et al., 1987b). Evidence from studies of SV40 DNA replication in uitro indicates the requirement of PCNA and suggests PCNA for coordinated leading and lagging strand synthesis at thereplication fork (Prelich et al., 1987b; Prelich and Stillman, 1988). These resultshave led to a proposal that polymerase 6 and a act as the leading and lagging strand DNA polymerases, respectively, at the replication fork (Downey et al., 1988; So and Downey, 1988; Blow,1988). DNA polymerases a and 6 have certain biochemical and physicochemical similarities. Both DNA polymerases are sensitive to inhibition by aphicicolin, which is an inhibitor for cellular DNA synthesis (Ikegami et al., 1978; Wang et al., 1984; Lee et al., 1984), and the catalytic polypeptide of both DNA polymerases have similar molecular mass and isoelectric points (Sedwick et al., 1972; Wonget al., 1986; Leeet ai.,1984; Lee and Toomey, 1987). However, DNA polymerases a and 6 have certain distinguishing biochemical properties, such as the chromatographic behavior, template-primer preferences, differential sensitivities to nucleotide analogs, associated enzymatic activities, i.e. primase for polymerase a and 3“s‘ exonuclease for polymerase 6, and the difference in response to the cell-cycle regulated protein(PCNA)(Byrnes et al., 1976; Wang et al., 1984; Byrnes, 1985; Lee et al., 1985; Wahl The abbreviations used are: PCNA, proliferating cell nuclear antigen; SDS, sodium dodecyl sulfate.

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DNA Polymerases a and 6 et al., 1986; Tan et al., 1986). The finding of a cryptic 3'-5' exonuclease associated with the catalytic polypeptide of Drosophila embryo DNA polymerase a, afterseparation from associated subunits(Cotterill etal., 1987), hasstimulated profound interestand speculation regarding the possible structural relationship between DNA polymerase a and 6 . Recently, a form of DNA polymerase 6 that is not stimulated by PCNA was purified from HeLa cells (Nishida et al., 1988; Syvaoja and Linn, 1989). This polymerase 6 is distinguished from the PCNA-dependent polymerase 6 for inherent reactivity on poly(A) .oligo(dT) (Syvaoja and Linn, 1989). In this report, we describe a comparison between polymerase a and the two forms of polymerase 6 (PCNA-dependent and PCNA-independent) from both cultured human cells and calf thymus tissue by immunological approach and detailed primary structural analysis by two-dimensional tryptic peptide mapping. An analysis of the relationship between the PCNAindependent form of polymerase 6 from HeLa cells and the PCNA-dependent form of polymerase 6 from calf thymus was also described. Our results demonstratethat theDNA polymerases a and 6 are immunologically and structurally distinct enzymes by these criteria and that the PCNA-independent human DNA polymerase 6 share some immunological and structural determinants with the PCNA-dependent calf thymus DNA polymerase 6.

5925

tion. Tryptic peptides of 100,000 cpm were analyzed on thin layer plates as described previously (Elder et al., 1977; Wong et al., 1986). The thinlayer plates were radioautographed on Kodak XAR-5 x-ray film at -70 "C with a Du Pont Lightening Plus intensifier screen. Control samples were performed by an identical procedure except that trypsin was omitted from the digestion reaction. RESULTS

Immunological Comparison of DNA Polymerases a and 6Monoclonal antibody specifically against DNA polymerase a, SJK132-20, has been demonstrated to be the most crossreactive among the panel of 16 monoclonal anti-DNA polymerase a antibodies (Tanaka et al., 1982; Miller et al., 1988). Monoclonal antibody SJK132-20 is able to neutralize DNA polymerase a activities as well as recognize a unique antigenic epitope displayed on the 165-180-kDa polymerase a polypeptides in cell lysates from phylogenetically diverse vertebrates (Miller et al., 1988). Monoclonal antibody SJK132-20 was thus selected to assess the cross-reactivity between DNA polymerase a and the PCNA-independent and the PCNAdependent DNA polymerase 6 from cultured human cells and from fetal calf thymus tissue, respectively. Neutralization assays indicate that this monoclonal antibody has no detectable reactivity with either form of DNA polymerase 6, but DNA polymerase a from either cultured human cells or calf thymus exhibits comparable sensitivity (Fig. 1). Conversely, a polyclonal antiserum raised against the PCNA-dependent MATERIALS AND METHODS calf thymus DNA polymerase 6 is able to neutralize the DNADNA Polymerases-Immunoaffinity purified human KB cell DNA polymerase a of specific activity 100,000-200,000 units/mg prepared synthesizing activity of both forms of polymerase 6 while as described (Wang et al., 1984; Wong et al., 1986) was from the having no effect on the DNA polymerase a activity from laboratory of T. Wang (StanfordUniversity); HeLa DNA polymerase either calf thymus or human KBcells (Fig. 2). 6, specific activity of 13,000-24,000 units/mg, prepared according to Identification of Polypeptide Displaying the Epitopes RecSyvaoja and Linn(1989) was from the laboratory of S. Linn (Univer- ognizedbyPolyclonal Antiserum against PCNA-dependent sity of California at Berkeley); immunoaffinity purified calf thymus Calf Thymus DNA Polymerase 6"Immunoblots were perDNA polymerase a of specific activity 35,000 units/mg (Chang et al., 1984) was from the laboratory of L. Chang (Uniformed Services formed on polymerase a purified from human KB and calf

University of the Health Sciences, Bethesda, MD); and fetal calf thymus tissue DNA polymerase 6 of specific activity 27,000 units/mg purified as described in Lee et al. (1984) was from the laboratory of A. G. So (University of Miami). The unitof each DNA polymerase is defined as the incorporation of 1 nmol of 2'-deoxynucleotide 5'monophosphate/h under the assay condition of the respective reports describing the purification procedures. Antibodies-Neutralizing monoclonal antibody against DNA polymerase a,SJK132-20, (Tanaka et al., 1982) was from the laboratory of T. Wang; murine polyclonal antiserum againstthe PCNA-dependent calfDNA polymerase 6 (Downey et al., 1988) was from the laboratory of A. G. So. Antibody Neutralization Assays-One-half of a unit of DNA polymerase a and PCNA-dependent calf thymus polymerase 6 and 0.1 unit of the PCNA-independent DNA polymerase 6 from HeLa cells were preincubated with varying amounts of antibody as indicated in the legend to Figs. 1 and 2 and described in Tanaka et al. (1982). The preincubation mixtures were then assayed for DNA polymerase a or 6 activity using poly(dA) .oligo(dT) (201) astemplate-primer (Tan et al., 1986). All except HeLa cell PCNA-independent DNA polymerase 6 were assayed in the presence of 1.6 pg/ml auxiliary protein (PCNA) from calf thymus tissue (Tan etal., 1986). SDS-Polyacrylamide Gel Electrophoresis and Immunoblots-Denaturing gel electrophoresis was performed as described (Laemmli, 1970)and stained with either CoomassieBrilliant Blue or ammoniacal silver as described (Wrayet al., 1981). For immunoblotting with polyclonal anti-calf polymerase 6 antibodies, proteins were separated by 8% SDS-polyacrylamide gel, transferred to nitrocellulose, probed with 5 p1 of primary antibody, and developed by using avidin-conjugated secondary antibody and biotin-conjugated peroxidase with diaminobenzidine chromophore as described in the Vectastain kit (Vector Laboratories, Inc.). Two-dimensional Peptide Mapping-The catalytic polypeptide for each DNA polymerase (Wong et al., 1986; Lee et al., 1984; Lee and Toomey, 1987) was isolated from a denaturing polyacrylamide gel after staining with Coomassie Brilliant Blue. The individual polypeptides were subsequently treated by radioiodination and trypsin diges-

50

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CALF THYMUS DNA POI

PERCENT ACTIVITY

0 150

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i

1

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b

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CALF THYMUS DNA pol

HUMAN DNA pol

I cI(

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b I o (

SJK 1 3 2 - 2 0 IgG (ygiassay)

FIG. 1. Effect of human DNA polymerase (Y monoclonal antibody SJKl32-20on DNA polymerase a and 6 activities. Antibody neutralization assays of each purified DNA polymerases were performed as described under "Materials and Methods" with the indicated amounts of monoclonal antibody SJK132-20. The data are shown as the percentage of activity remaining as compared with no antibody in the preincubations. A, activity with preimmune monoclonal P3 IgG, A, activity remaining with monoclonal antibody SJK132-20. Percentage of activity for each polymerase is defined as pmol of 2'-deoxynucleoside 5'-monophosphate incorporated per 15 min at 37 'C, except for the PCNA-independent HeLa polymerase 6 at 30 "C. One hundred percent is 30 pmol for calf polymerase a,30 pmol for human polymerase a,65 pmol for the PCNA-dependent calf polymerase 6, and 25 pmol for the PCNA-independent HeLa polymerase 6.

DNA Polymerases a and 6

5926

polymerase 6 activity (Fig. 2) and to recognize epitope(s) on a specific polypeptide of 130 kDa (Fig. 3B) suggests that the catalytic activityof calf thymus DNA polymerase 6 resides in this polypeptide. The inability of this serum to identify any antigenicepitopeon polypeptide components from either human or calf DNA polymerase a enzyme fraction further substantiates the conclusion from Figs. 1 and 2 that DNA polymerases a and 6 are immunologically distinct. Comparative Analysis of Primary Structure of the Catalytic Polypeptides of DNA Polymerase a and the PCNA-dependent Calf Polymerase 6"Assessment of the structuralrelationships between DNA polymerase a and the PCNA-dependent calf polymerase 6 was carried out by two-dimensional tryptic 50 peptidemapping of their catalytic polypeptides. Previous HUMAN DNA pol o( CALF THYMUS DNA pol o( experiments have documented that the catalytic polypeptide 0 I I I I ' I I I I I I I I I I I of DNA polymerase a from either human orcalf thymus isa 0 1 2 3 0 1 2 3 cluster of polypeptides predominantly of 180 and 165 kDa MOUSE ANTI-CALF THYMUS DNA pol b PI) demonstrated in vitroby peptide mappingto be derivatives of FIG.2. Effect of murine polyclonal antisera against calf thymus DNA polymerase 6 on DNA polymerase a and 8 activ- the same primary structure (Wong et al., 1986; Chang et ul., ities. Assays were performed as described in Fig. 1 and under "Ma- 1984). Comparison of tryptic peptides of the 130-kDa polyterials and Methods" with the indicated amounts of mouse polyclonal peptide of the PCNA-dependent calf DNA polymerase 6 with as the percentage the 165-kDa polymerase a catalytic polypeptides from calf anti-calf polymerase 6 antisera. The data are shown of activity remaining as compared with no antibody in the preincuthymus and human KBcells (Fig. 4, A-C) demonstrates that 0, activity bations. 0, activity remaining with preimmune serum; the catalytic polypeptides of DNA polymerase a from calf remaining with mouse polyclonal anti-polymerase 6 serum. Percentthymus and human KB cells and the PCNA-dependent calf age of activity is definedas in Fig. 1. One hundred percent is60 pmol for calf thymus polymerase a,40 pmol for the PCNA-dependent calf thymus polymerase 6 have distinct and unrelatedpeptide thymus polymerase 6,60pmol for human polymerase a,and 25 pmol maps, whereas polymerase a from calf and human have simfor the PCNA-independent HeLapolymerase 6. ilar, although not absolutely identical, two-dimensional peptide patterns (Fig. 4, A and C). This latter result further supports our previous finding that two restriction fragments Calf DNA Human DNA of human DNA polymerase a cDNA sequences, which compolymerase polymerase prise five of the six consensus sequences, are capable of cross6 a 6 a hybridizing calf genomic DNA (Miller et ul., 1988). Structural Analysis of PCNA-independent Human PolymA B C D E F O H erase 6"Biochemical evidence corroborated by physical measurements has implied that the predominant>200-kDa poly150

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CALFTHYMUSDNApolb

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HUMANDNApol b

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FIG.3. Immunoblot of DNA polymerase

and 6 fractions from both calf thymus and cultured human cells. The immu-

4

a

noblot was performed as described under "Materials and Methods" with 300 ng of each DNA polymerase enzyme fraction. The specific a silver immunoblot of each polymerase fraction is shown along with stain of the protein componentsof SDS gel of the respective polymerase fraction. A, C, E , and C represent the silver stain of SDS gel of respective polymerases. B, D, F, a n d H a r eimmunoblots.

thymus and the two forms of polymerase 6 and compared with a silver-stained SDS gelof each polymerase fraction used (Fig. 3). The antiserum recognizes a unique epitope displayed exclusivelyon a130-kDa polypeptide of the PCNA-dependent calf thymus DNA polymerase 6 enzyme fraction (Fig. 3). Surprisingly, despite the ability of this polyclonal serum to neutralize the purified PCNA-independent HeLa polymerase 6 enzymatic activity (Fig. 2), it does not reveal any antigenic polypeptides in the HeLa DNA polymerase 6 enzyme fraction (Fig. 3). None of the polypeptides in DNA polymerase a preparations from either calf thymus tissue or human KB cells demonstrates anyantigenic epitoperecognizable by this anti-polymerase 6 serum. The ability of the polyclonal antibodies to neutralize calf

WDNA

FIG.4. Comparison of primary structureby tryptic peptide map on the catalyticpolypeptides of DNA polymerases a and 6 from calf thymus and of cultured human cells. The procedure wasperformed as described under "Materials and Methods," and 100,000cpm of each tryptic peptide was analyzed on thin layer plates. Tryptic peptides of similar migration among thoseof PCNA-dependent calf thymus ( B ) and PCNA-dependent HeLa polymerase b (D) are indicatedby arrows. Tryptic peptidesof similar migrationbetween human polymerase a (C)and b (D)are marked by a dotted box.

DNA Polymerases cy and 6 peptide of the PCNA-independent HeLapolymerase 6 enzyme fraction iscoincident with the polymerase 6 enzymatic activity (Syvaoja and Linn, 1989). Thus, it is of interest to compare the tryptic peptide map of the >200-kDa predominant polypeptide with the 130-kDa polypeptide of the PCNA-dependent calf polymerase 6 and human polymerase a (Fig. 4, D to B and C). Two-dimensional tryptic peptide mapping analysis of this >200-kDa polypeptide of the PCNA-independent human polymerase 6 (Fig. 4 0 ) demonstrates ageneral dissimilar peptide pattern from that of the PCNA-dependent calf polymerase 6 polypeptide (130 kDa; Fig. 4B) and from the catalytic polypeptide of human KB polymerase (Y (180- or 165-kDa polypeptides; Fig. 4C). However, several similar tryptic peptide spots canbe correlated between this PCNA-independent HeLa polymerase 6 polypeptide and thecatalytic polypeptides of PCNA-dependent calf polymerase 6 (indicated by arrows in Fig. 4, B and D )and the human polymerase (Y (boned in dotted lines in Fig. 4, C and D ) . DISCUSSION

The requirement of PCNA for efficient SV40 DNA replication in vitro and for the coordination of leading and lagging strand synthesis and the finding that PCNA is an auxiliary factor selectively affecting only DNA polymerase 6 enzymatic activity suggest that DNA polymerase 6, like polymerase a, plays an essential biologicalrole inthe eukaryotic DNA replication process (Tan et al., 1986; Prelich et al., 1987a, 1987b; Bravo et al.,1987; Prelich and Stillman, 1988; Downey et al., 1988;Wold et al., 1988). However, complete in uitro formation of RFI’ SV40 DNA was reported with DNA containing SV40 origin, large T antigen, andsix protein fractions purified from HeLa cells without the requirement of polymerase 6 or PCNA (Wobbe et al., 1987; Dean et al., 1988). Furthermore, a distinct form of polymerase 6 purified from cultured humancells or calf thymus tissueby two independent laboratories hasbeen demonstrated to be PCNA-independent (Savaoja and Linn, 1989; Sabatino et al., 1988; Bambara et al., 1989). These findings, in addition to the finding of an intrinsic cryptic exonuclease in the catalytic polypeptide of Drosophila embryo polymerase a (Cotterill et al., 1987), indicate that it is imperative to clarify the relationship between polymerases (Y and 6 and the relationship of the two forms of DNA polymerase 6 . In thisreport, we have demonstrated that DNA polymerases (Y and 6 display reciprocally exclusive epitopes recognized by respective specific antibodies and that their catalytic subunits have distinct tryptic peptide maps. The recent identification of a form of DNA polymerase 6 that is not stimulated by PCNA adds more complexity to the replicative polymerases involved in DNA replications. The relationship between the PCNA-dependent and PCNA-independent forms of polymerase 6 is presently unclear but intriguing. The PCNA insensitivity of the two polymerase 6 preparations has been demonstrated not to result from the presence of contaminating PCNA in theenzyme preparations (Syvaoja and Linn, 1989; Bambara et al., 1989). In the HeLa polymerase 6 preparation, there is neither detectable 36-kDa PCNA polypeptide nor any measurable effect of anti-PCNA antibody on its reactivity with poly(dA) .oligo(dT) (Syvaoja and Linn, 1989). Immunoblot analysis with anti-PCNA antibody also failed to detect the presence of PCNA in thePCNAindependent calf thymus polymerase 6 preparation (So and Downey,1988; Bambara et al., 1989). However, both the humanand calf PCNA-independent polymerase 6 can be neutralized by the polyclonal serum specifically raised against the PCNA-dependent calf thymus polymerase 6 antigen (So and Downey, 1988, and Fig. 2).

5927

The present finding that thepolyclonal antiserum prepared against the PCNA-dependent calf thymus polymerase 6 neutralizes the activity of the PCNA-independent HeLa polymerase 6 but does not immunoblot any polypeptides in this preparation could have several explanations. It is possible that theepitopes of the PCNA-independent HeLa polymerase 6 recognizedby the polyclonal serum in the neutralizing enzyme assay areinanative condition, underdenatured immunoblotting, and that the epitopes may no longer be recognizableby the antibodies. The polymerase 6 preparations from calf thymus (thePCNA-dependent form) and from HeLa cells (the PCNA-independent form) used in this study have comparable specific activities, i.e. 27,000 units/mg for calf thymus enzyme and 24,000 units/mg for HeLa enzyme. The immunoblot was performed with equal units of polymerase 6 enzyme activity, i.e. with comparable amounts of protein from each polymerase 6 enzyme fraction. If neither of the two predominant protein components of the HeLa polymerase 6 preparation is the catalytic protein, this PCNA-independent HeLa polymerase 6 will be several orders of magnitude more active than the PCNA-dependent calf polymerase 6 in uitro. The specific activity value of this PCNA-independent HeLa polymerase 6 supports the notion that the>200-kDa polypeptide of this HeLa enzyme fraction corresponds to thecatalytic function of polymerase 6 activity. Furthermore, the polymerase 6 activity sediments at 11.1 S in low salt and6.8 S in high salt. In both cases, polymerase activity cosediments with the major visible polypeptide of >ZOO kDa (Syvaoja and Linn, 1989). Alternatively, it is also possible that the antibody species inthis polyclonal serum, which neutralizes both PCNA-independent polymerase 6 from calf thymus andHeLa cells, is different from the antibody species that immunoblots the 130-kDa polypeptide in the PCNA-dependent calf thymus polymerase 6 preparation. The tryptic peptide mapping experiments shown in Fig. 4 indicate that polymerase (Y and the two forms of polymerase 6 have distinct primary structures. The PCNA-dependent calf polymerase 6 catalytic polypeptide (130 kDa) and the PCNAindependent HeLa polymerase 6 polypeptide (>ZOO kDa), in general, have dissimilar tryptic peptide patterns; however, several common tryptic peptides do exist. A few tryptic peptide spots of similar migration between human polymerase (Y and the PCNA-independent human polymerase 6 can also be identified. Whether these few similar tryptic peptides represent the limited primary sequence similarity between these polymerases or just a statistically fortuitous coincidence is unknown at present. Circumstantial evidence implicates the PCNA-dependent polymerase 6 in DNA replication. Although the PCNA-independent polymerase 6 from HeLa cells has been shown to be required for DNA repair synthesis in xeroderma pigmentosum human fibroblasts (Nishida et al., 1988), a role of this polymerase 6 species in DNA replication should not be ruled out at present. The resolution of the relationship of the two forms of polymerase 6 awaits further characterization andcomparison of polymerase 6 purified by the three defined procedures from both HeLa cells and calf thymus tissues (Crute et al., 1986; Syvaoja and Linn, 1989; Leeet al., 1984). The identification of several consensus regions in the primary protein sequence of human DNA polymerase (Y having similarity to both prokaryotic and eukaryotic replicative DNA polymerases (Wong et al., 1988; Wang e t at., 1989), and the finding of evolutionary conservation of these sequences in replicative polymerases from phylogenetically distinct species (Miller e t a i , 1988) strongly suggest that these DNA polymerases evolved from a common ancestral gene and also imply

DNA Polymerases a and 6

5928

Gibbs, J. S., Chiou, H. C., Bastow, K. F., Cheng, Y.-C., and Coen, D. that these sequences are conserved to maintain elementary M. (1988) Proc. Natl. Acad. Sci. U. S. A. 8 5 , 6672-6676 butessential DNApolymerization functions.Primarysequence comparison of human DNA polymerase a and DNA Hammond, R. A., Byrnes, J. J., and Miller, M. R. (1987) Biochemistry 26,6817-6824 polymerase of herpes simplex virus mutants of altered sensi- Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H., and tivity to competitive inhibitors or analogs of 2’-deoxynucleoMano, Y. (1978) Nature 2 7 5 , 458-460 tide triphosphate, such as aphidicolin and several antiviral Laemmli, U. K. (1970) Nature 227,680-685 drugs, has provisionally assigned two of the consensus se- Larder, B. A., Kemp, S. D., and Darby, G. (1987) EMBO J. 6 , 169175 quences directly participating in 2’-deoxynucleotide triphosM. Y. W. T., and Toomey, N. L. (1987) Biochemistry 26,1076phate binding and PPi hydrolysis (Gibbs et al., 1985, 1988; Lee, 1085 Larder et al., 1987; Tsurumi et al., 1987; Wong et al., 1988; Lee, M. Y. W. T., Tan, C.-K., Downey, K. M., and So, A. G. (1981) a and 6 exhibit Wang et al., 1989).Becausepolymerases Prog. Nucleic Acid Res. Mol. Biol. 2 6 , 88-96 similar extent of competitive inhibition by aphidicolin and Lee, M. Y. W. T., Tan, C.-K., Downey, K. M., and So, A. G. (1984) Biochemistry 23,1906-1913 similar DNA polymerization mechanisms, the two polymerLee, M. Y. W. T., Toomey, N. L., and Wright, G. E. (1985) Nucleic ases may containsimilar or identicalstructuralelements Res. 13,8623-8630 involving in substrate (2’-deoxynucleotide triphosphate) rec- Li,Acids J. J., and Kelly, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, ognition and interaction. It is attractive to speculate that 6973-6977 polymerase 6 , like polymerase a , also contains these consensusLi, J. J., and Kelly, T. J. (1985) Mol. Cell. Biol. 5 , 1238-1246 regions in its primary protein sequence. The findingof distinct Mathews, M. B., Bernstein, R. M., Franza, B. R., and Garrels, J. I. (1984) Nature 309,374-376 primary structures by the tryptic peptide mapping of these two DNA polymerases argues against this possibility; how- Miller, M. A.. Korn,. D... and Wang, T. S.-F. (1988) Nucleic Acids Res. 16,’7961-7973 ever, this possibility may not be entirely ruled out by compar- Miller. M. R.. Ulrich, R. G.. Wana. -. T. S.-F., and Korn, D. (1985) J. ative tryptic peptide mapping. Resolution of this interesting ~ i o Chem.’ i 26o,i34-138 question awaits the future isolation of polymerase 6 cDNA Miller, M. R., Seighman, C., and Ulrich, R. G . (1986) Biochemistry 24,7440-7445 and comparative analysis of the deduced amino acid sequence with polymerase a and other prokaryotic or eukaryotic repli- Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U. S. A. 83,2869-2873 cative polymerases. Murakami, Y., Wobbe, C. R., Weissbach, L., Dean, F. B., and Hunvitz, Acknowledgments-We thank Drs. Andrew Holmes and Lucy Chang for their generous gift of the immunoaffinity purified calf thymus DNA polymerase a and Drs. Robert Lehman and Alan Wahl of Stanford University for their helpful discussions and critical comments on the manuscript. REFERENCES Bamhara, R. A,, Myers, T. W., and Sabatino, R. D. (1989) in The Eukaryotic Nucleus: Molecular Structure and Macromolecular Assemblies (Straus, p., andWilson, s.,eds) Telford Press, New York, in press Blow, J. J. (1988) Bioessays 8 , 149-152 Bravo, R. (1986) Exp. Cell Res. 1 6 3 , 287-293 Bravo, R., and Celis, J. E. (1980) J. Cell Biol. 84, 795-802 Bravo, R., Frank, R., Blundell, P. A., and Macdonald-Bravo, H. (1987) Nature 326,515-517 Byrnes, J. J. (1985) Biochem. Biophys. Res. Commun. 132,628-634 Bvrnes. J. J.. Downey, K. J., Black, V. L., and So, A. G. (1976) “Biochemist& 1 5 , 2817-2833 Chane. L. M. S.. Rafter. E.. Aunl. - . C., and Bollum, F. J. (1984) J . Biol. Chlm. 2 5 9 , 14679-14687 Cotterill, S. M., Reyland, M. E., Loeb, L. A., and Lehman, I. R. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 5653-5639 Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry ~~

I

25,26-36

Dean, F. B., Dodson, M., Borowiec, J. A., Ishimi, I., Goetz, G. S., Bullock, P.,Matson, S. W., Echols, H., and Hunvitz, J. (1988) Cancer Cells 6 , 113-122 Decker, R. S., Yamaguchi, M., Rossenti,P., Bradley, M.K., and DePamphilis, M. L. (1987) J. Biol. Chem. 2 6 2 , 10863-10872 Downey, K. M., Tan, C.-K., Andrews, D. M., Li, X., and So, A. G. (1988) Cancer Cells 6 , 403-410 Dresler, S. L., and Frattini,M. G. (1986) Nucleic Acids Res. 14,70937102

Elder, J. H., Pickett, R. A., 11, Hampton, J., and Lerner, R. A. (1977) J . Biol. Chem. 252,6510-6515 Fry, M., and Loeb, L. A. (1986) Animal Cell DNA Polymerases, CRC Press, Boca Raton, FL Gibbs, J. S., Chiou, H. C., Hall, J. D., Mount, D. W., Retondo, M. J., Weller, S. K., and Coen, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,7969-7973

J. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 2869-2873 Nishida, C., Reinhard, P., and Linn, S. (1988) J. Biol. Chem. 263, 501-510

Prelich, G., and Stillman, B. (1988) Cell 5 3 , 117-126 Prelich,G., Kostura, M., Marshak, D. R., Mathews, M. B., and Stillman, B. (1987a) Nature 326,471-475 Prelich, G., Tan, C.-K., Kostura, M., Mathews, M.B., So, A. G., Downey, K. M., and Stillman, B. (1987) Nature 3 2 6 , 517-520 Sabatino, R. D., and Bambara, R. A. (1988) Biochemistry 2 7 , 22662271

Sedwick. W. D.. Wane. T. S.-F.. and Korn, D. (1972) J. Biol. Chem. Yl

247,5026-5033

So, A. G., and Downey, K. M. (1988) Biochemistry 27,4591-4595 Stillman. B.W.. and Gluzman, Y. (1985) Mol. Cell. Biol. 5 , 20512060

Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 2 6 4 , 2489-2497 Tan, C.-K., Castillo, C., So, A. G., and Downey, K. M. (1986) J. Biol. Chem. 2 6 1 , 12310-12316 Tanaka, S., Hu, S.-Z., Wang, T. S.-F., and Korn, D. (1982) J. Biol. Chem. 257,8386-8390 Tsurumi, T., Maeno, K., and Nishiyama, Y. (1987) J. Virol. 61,388394

Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., and Bambara, R. A. (1986) Biochemistry 25,7821-7827 Wahl, A. F., Geis, A. M., Spain, B. H., Wong, S. W., Korn, D., and ‘Wang, T. S.-F. (1988) Mol. Cell. Biol. 8 , 5016-5025 Wane. T. S.-F.., Hu.. S.-Z.. and Korn. D. (1984) J. Biol. Chem. 2 5 9 , 1854-1865

Wang, T. S.-F., Wong, S. W., and Korn, D. (1989) FASEB J., 3 , 1421

Wobbe, C.R., Weissbach, L. J., Borowiec, J. A., Dean, F. B., Mura Kami, Y., Bullock, P., and Hunvitz, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,1834-1838 Wold, M. S., Li, J. J., Weinberg, D. H., Virshup, D. M., Sherley, J. M., Verheyen, E., and Kelly, T. (1988) Cancer Cells 6 , 133-142 Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J . BioE. Chem. 261,7958-7968 Wong, S. W., Wahl, A. F., Yuan, P.-M., Arai, N., Pearson, B. E., Arai, K-I., Korn, D., Hunkapiller, M. W., and Wang, T. S.-F. (1988) EMBO J. 7,37-47 Wray, W., Boulikas, T., Wray, V. P., and Hancock, K. R. (1981) Anal. Biochem. 1 1 8 , 197-203